Development of an organ on chip microfluidic platform with real-time viability sensing capabilities

Abstract

Estimated cost per drug is over $1.5 billion, with success rates ranging between 3 and 14%. Current models used in preclinical trials are based in static in vitro and animal models, which fail to recapitulate complex environments from the human body. The organ-on-chip model emerged middle way between both aforementioned models, providing high experimental throughput while maintaining a complex environment for drug testing. In this thesis the design, development and fabrication of a compact and easy to use organ-on-chip platform was described. The platform is capable of cell culture with constant cell media perfusion underneath the cell culture, housed in individual microfluidic chips. Continuous epithelial barrier integrity assessment was possible via electrical impedance spectroscopy measurement using electrodes located underneath the cell culture. Fluidics were provided to each microfluidic chip individually, with placement, alignment and replacement possible using magnets in a ”plug-andplay” manner. Transepithelial resistance equivalent and cell barrier capacitance values were extrapolated from impedance spectroscopy data utilising an electrical circuit model. Complex non-linear square fit and Nelder-Mead minimisation algorithms were used to extrapolate cell media resistance, electrode parameters (electrode polarization) and cell barrier resistance (TER) and capacitance from the complex impedance spectroscopy data. Two different types of epithelial cell lines (bronchial and gut) were successfully cultured in the platform, with continuous transepithelial resistance (TER) and cell barrier capacitance measurements throughout the experiments. Both cell lines displayed lower TER comparatively to static models (Transwells), with results matching recent findings in literature regarding TER of cell cultures with and without flow. Additionally, epithelial barrier integrity disruption was measured following Triton X-100 or viral mimetic apical stimulation using human bronchial epithelial cells, with both conditions displaying a decrease in barrier integrity after stimulation. Lastly, a droplet generator was designed, developed and used to compartmentalise cell media from the organ-on-chip platform, allowing temporal analysis of epithelial barrier molecular permeability and/or excreted analytes. The droplet generator was composed of polydimethylsiloxane (PDMS) and was could generate droplets every 20 minutes in 8 channels simultaneously, with an average droplet volume of 9.47 ± 0.6 µL. Fluorescein diffusion across the porous membrane was assessed, with results similar to simulated data using COMSOL. Fluorescein-Dextran permeability across human bronchial epithelial cells following barrier formation was assessed with and without Triton X-100 apical stimulation. Both permeability and TER data reached the same conclusion, with Triton X-100 cultures displaying a decline in barrier integrity compared to media controls. Interleukin-8 production was assessed using the same cell line following apical stimulation with a viral mimetic, however no conclusions could be inferred due to data variability between repeats.

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Identifiers

ORCID for Hywel Morgan: orcid.org/0000-0003-4850-5676

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